The continuing search for new and effective antibacterial agents that can treat infections caused by organisms that are increasingly resistant to known classes of antibacterial agents has identified a plethora of potential next generation antibiotics. Many of these agents have subsequently been shown to demonstrate either poor physiochemical properties, an increased tendency to induce bacterial resistance, poor toxicological profiles, or low efficacy in vivo. Over the past decade or so, certain antibacterial peptides and glycopeptides isolated from insects have been noted as promising candidates for drug development programs (see, e.g., Hultmark, Trends Genet., 9:178-183 (1993); Gillespie et al., Annu. Rev. Entomol., 42:611-6443 (1997); Otvos, Jr. et al., Protein Science 9:742 (2000); International Patent Publication No. WO 94/05787, published Mar. 17, 1994; French Patent No. 2733237, granted Oct. 25, 1996; International Patent Publication No. WO 99/05270, published Feb. 4, 1999; International Patent Publication No. WO 97/30082, published Aug. 21, 1997; French Patent No. 2695392, granted Mar. 11, 1994; French Patent No. 2732345, granted Oct. 4, 1996; and International Patent Publication No. WO 00/78956, published Dec. 28, 2000.)
While many antibacterial peptides from other origins kill bacteria by disrupting the cell membrane or cell wall of the bacteria, a subset of the insect-derived antibacterial peptides have an unusual mode of action, i.e., they inhibit the bacterial chaperone protein DnaK. Two such peptides are drosocin, a 19 amino acid residue peptide from Drosophila (Bulet et al., J. Biol. Chem. 268:14893-14897 (1993)) and pyrrhocoricin, a 20 amino acid residue peptide from Pyrrhocoris (Cociancich et al, Biochem. J. 300:567-575 (1994)). Drosocin and pyrrhocoricin are glycopeptides characterized by the presence of a disaccharide in the mid-chain position. The presence of the disaccharide increases the in vitro antibacterial activity of drosocin, but decreases the activity of pyrrhocoricin (Bulet et al., supra; Hoffmann et al., Biochim. Biophys. Acta, 1426:459-467 (1999)).
While active in vitro, both drosocin and pyrrhocoricin are known to be highly susceptible to proteolytic degradation in the presence of mammalian serum. Both aminopeptidase and carboxypeptidase cleavage products are observed. Metabolites lacking as few as five amino terminal or two carboxy terminal amino acids have been shown to be inactive as antibacterial agents in vitro (Bulet et al., supra; Hoffmann et al., supra).
The interaction of non-glycosylated pyrrhocoricin with bacterial DnaK has been extensively studied (see, e.g., Otvos, Jr. et al, Biochemistry 39:14150-14159 (2000); Kragol et al., Biochemistry 40:3016-3026 (2001)). Residues within the N-terminal half of the peptide have been implicated in binding to DnaK, and were shown to specifically interact with helices D and E of the helical lid of the bacterial protein (Kragol et al., Eur. J. Biochem. 269:4226-4237 (2002)).
DnaK has been demonstrated to be the central protein in a multiprotein bacterial chaperone system including the chaperone protein DnaK and a variety of co-chaperone proteins such as DnaJ and GrpE. The co-chaperone proteins are essential to the efficient physiological processing of both natural and unnatural substrates. One role for this chaperone system is to catalyze the refolding of either unfolded or misfolded bacterial proteins, as is evident from the role of this system in the heat-shock response. An additional role of the DnaK chaperone system is the regulation of gene expression through the processing of specific RNA polymerase subunits.
DnaK deletion mutants of many organisms have been generated and overall the mutant strains have been shown to exhibit lower growth rates, greater susceptibility to environmental stress, reduced viability in cellular environments, and reduced ability to establish infections in vivo compared to the wild-type strains.
The susceptibility of DnaK deletion mutants to known antibacterial agents has been only briefly examined. Wolska et al. (Microb. Drug Resist. 6:119-126 (2000)) have shown that an E. coli DnaJ deletion mutant and the corresponding DnaK/DnaJ double deletion mutant were not more susceptible to ampicillin, chloramphenicol or tetracycline under routine conditions. Conversely, Yamaguchi et al. (BMC Microbiol. 3:16 (2003)) demonstrated a moderate increase in susceptibility of an E. coli DnaK deletion mutant to levofloxacin.
While the role of the DnaK chaperone system in bacterial growth and survival is only now starting to be appreciated and understood, the role and utility of DnaK inhibitors as antibacterial agents is not well established. Accordingly, there is a need to investigate and exploit the useful therapeutic activities of DnaK inhibitors, including pyrrhocoricin, drosocin and their analogs.